Mechanism for acivicin inactivation of triad glutamine amidotransferases

Acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid] was investigated as an inhibitor of the triad glutamine amidotransferases, IGP synthase and GMP synthetase. Nucleophilic substitution of the chlorine atom in acivicin results in the formation of an imine-thioether adduct at the active site cysteine. Cys 77 was identified as the site of modification in the heterodimeric IGPS from Escherichia coli (HisHF) by tryptic digest and FABMS. Distinctions in the glutaminase domains of IGPS from E. coli, the bifunctional protein from Saccharomyces cerevisiae (HIS7), and E. coli GMPS were revealed by the differential rates of inactivation. While the ammonia-dependent turnover was unaffected by acivicin, the glutamine-dependent reaction was inhibited with unit stoichiometry. In analogy to the conditional glutaminase activity seen in IGPS and GMPS, the rates of inactivation were accelerated > or =25-fold when a nucleotide substrate (or analogue) was present. The specificity (k(inact)/K(i)app) for acivicin is on the same order of magnitude as the natural substrate glutamine in all three enzymes. The (alphaS,5R) diastereomer of acivicin was tested under identical conditions as acivicin and showed little inhibitory effect on the enzymes indicating that acivicin binds in the glutamine reactive site in a specific conformation. The data indicate that acivicin undergoes a glutamine amidotransferase mechanism-based covalent bond formation in the presence of nucleotide substrates or products. Acivicin and its (alphaS,5R) diastereomer were modeled in the glutaminase active site of GMPS and CPS to confirm that the binding orientation of the dihydroisoxazole ring is identical in all three triad glutamine amidotransferases. Stabilization of the imine-thioether intermediate by the oxyanion hole in triad glutamine amidotransferases appears to confer the high degree of specificity for acivicin inhibition and relates to a common mechanism for inactivation.     

Inhibition of gamma-glutamyl transpeptidase decreases amino acid uptake in human keratinocytes in culture

Acivicin inhibits gamma-glutamyl transpeptidase activity in human keratinocytes in culture. Treatment of these cells with acivicin produces a decrease in the uptake of L-[U-14C]alanine, 2-amino-[1-14C]-isobutyrate, L-[U-14C]leucine and 1-aminocyclopentane-1-[14C]carboxylate. D-[U-14C]glucose uptake is not affected by the presence of acivicin. These results support, for the first time in vitro, the hypothesis that the gamma-glutamyl cycle may be involved in amino acid uptake by human cells.     

Inhibition of the hydrolytic and transpeptidase activities of rat kidney gamma-glutamyl transpeptidase by specific monoclonal antibodies.

Monoclonal antibodies (mAb) against the native form of rat kidney gamma-glutamyl transpeptidase (GGT) were isolated by screening hybridomas with rat kidney brush-border membrane vesicles. They were directed against protein rather than sugar epitopes in that each recognized all GGT isoforms. All of them inhibited partially the enzyme activity of GGT. They were specific in that they inhibited the rat enzyme, but not the mouse or human enzyme. Kinetic analyses were carried out with free GGT and GGT-mAb complexes with d-gamma-glutamyl-p-nitroanilide in the presence or absence of maleate, or in the presence or absence of alanine, cysteine, cystine or glycylglycine as gamma-glutamyl acceptors. mAbs 2A10 and 2E9 inhibited the hydrolytic and glutaminase activities of GGT and had little effect on the transpeptidation activity of the enzyme, whereas mAbs 4D7 and 5F10 inhibited transpeptidation, but not hydrolytic or glutaminase activities. mAb 5F10 mimicked the effect of maleate on GGT, in that it inhibited transpeptidation, enhanced the glutaminase activity and increased the affinity of the donor site of GGT for acivicin. Such mAbs may be useful for long-term studies in tissue cultures and in vivo, and for the identification of GGT epitopes that are important for the hydrolytic and transpeptidase activities.     

Glutaminase in neurons and astrocytes cultured from mouse brain: Kinetic properties and effects of phosphate,glutamate, and ammonia

Phosphate activated glutaminase comprises two kinetically distinguishable enzyme forms in cultures of cerebellar granule cells, of cortical neurons and of astrocytes. Specific activity of glutaminase is higher in cultured neurons compared with astrocytes. Glutaminase is activated by phosphate in all cell types investigated, however, glutaminase in astrocytes reguires a much higher concentration of phosphate for half maximal activation. One of the products, glutamate, inhibits the enzyme strongly, whereas the other product ammonia has only a slight inhibitory action on the enzyme.     

Uptake and metabolism of glutamine in cultured kidney cells

The metabolism of glutamine was investigated in cultured rat kidney cells. Glutamine utilization and product formation were followed as a function of time at either 10 microM or 1 mM initial glutamine concentration. At 1 mM glutamine, glutamate and gamma-glutamylglutamate were the major products formed at the end of a 5-min incubation period; glutamate accounted for 46% while gamma-glutamylglutamate accounted for 33% of the glutamine utilized. With time, glutamate continued to accumulate while gamma-glutamyl peptide formation leveled off. The role of gamma-glutamyl transpeptidase was assessed by using hippurate, a physiological activator of gamma-glutamyl transpeptidase and acivicin, L-(alpha S,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, an inhibitor of gamma-glutamyl transpeptidase. Hippurate, 4 mM, increased the utilization of glutamine and the formation of glutamate, gamma-glutamyl peptides and ammonia. Exposure of cells to acivicin resulted in 98% inhibition of gamma-glutamyl transpeptidase without effecting phosphate-dependent glutaminase activity. Acivicin inhibition resulted in a decreased utilization of glutamine and product formation as compared to control; 5-oxoproline appearance fell 70%. The fractional distribution of glutamine carbon and nitrogen into its metabolic products in control, hippurate and acivicin-treated cells showed no change at the end of 60 min. The data provide evidence that gamma-glutamyl transpeptidase utilizes glutamine and forms gamma-glutamyl peptides in cultured kidney cells.     

Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness

African sleeping sickness is a fatal disease caused by two parasite subspecies: Trypanosoma brucei gambiense and T. b. rhodesiense. We previously reported that trypanosomes have extraordinary low CTP pools compared with mammalian cells. Trypanosomes also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. In this study, we have expressed and purified recombinant T. brucei CTP synthetase. The enzyme has a higher K(m) value for UTP than the mammalian CTP synthetase, which in combination with a lower UTP pool may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase is irreversibly inhibited by the glutamine analogue acivicin, a drug extensively tested as an antitumor agent. There is a rapid uptake of acivicin in mice both given intraperitoneally and orally by gavage. Daily injection of acivicin in trypanosome-infected mice suppressed the infection up to one month without any significant loss of weight. Experiments with cultured bloodstream T. brucei showed that acivicin is trypanocidal if present at 1 mum concentration for at least 4 days. Therefore, acivicin may qualify as a drug with "desirable" properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi.     

Inhibitory effect of interferons and tumor necrosis factor on syncytium formation by bovine leukemia virus

The treatments of the bovine spleen cells BESp with recombinant human leukocyte interferon (HuIFN-alpha A) and human fibroblast interferon (HuIFN-beta) inhibit the syncytium formation by bovine leukemia virus (BLV) and the virus yield, depending on the IFN doses. But HuIFN-gamma did not inhibit the syncytium formation. The tumor-necrosis factor (TNF) also inhibits the syncytium formation and the virus yield, depending on the TNF doses. Furthermore, the combination of the HuIFN-alpha A with the TNF inhibits the syncytium formation more strongly than the HuIFN-alpha A alone or the TNF alone.     

A new acivicin prodrug designed for tumor-targeted delivery

Acivicin is an antitumor agent known to inhibit cell growth. A new prodrug 9b of acivicin 10 was synthesized, based on a p-hydroxybenzylcarbamate self-immolative spacer capable to release acivicin under esterase activity. The prodrug includes a maleimide-containing arm for linkage with thiol-containing macromolecules such as antibodies. This molecule is intended for the conception of bioconjugates to target an inactive acivicin precursor to tumor cells, when linked to a monoclonal antibody (mAb) which recognizes a tumor-specific antigen. Prodrug cleavage by plasmatic esterases will then restore the acivicin's activity toward tumor cells. We report here the synthesis and the in vitro characteristics of the prodrug. As expected, its inhibitory activity against the gamma-glutamyl transpeptidase (gamma-GT) enzyme and its cytotoxicity towards HL-60 cells were highly reduced compared to the parent drug. The chemical and plasmatic hydrolysis kinetics of the compound was studied by HPLC. The prodrug is stable, being slowly hydrolyzed in pH 7.6 buffer at 37 degrees C with a half-life of 37 h. It is converted into an active acivicin under the effect of pig liver esterase, and its half-life in human plasma is 3 h. These results indicate this compound may be further used as a prodrug-antibody conjugate, to target acivicin to malignant cells.     

Glutamine synthetase and glutaminase activities in various hepatoma cells

Glutamine synthetase and glutaminase activities in a series of hepatoma cells of human and rat origins were determined for comparison with normal liver tissues. Marked decrease in glutamine synthetase activity was observed in the tumor cells. Phosphate-dependent and phosphate-independent glutaminase activities were increased compared with those from normal liver tissues. Well coupled mitochondria were isolated from HuH 13 line of human hepatoma cells and human liver. Oxypolarographic tests showed that glutamine oxidation was prominent in the tumor mitochondria, while mitochondria from the liver showed a feeble glutamine oxidation. Glutamine oxidation was inhibited by prior incubation of the mitochondria with DON (6-diazo-5-oxo-L-norleucine), which inhibited mitochondrial glutaminase. These results indicate that the product of glutamine hydrolysis, glutamate, is catabolized in the tumor mitochondria to supply ATP.     

Mixture design as first step for improved glutaminase production in solid-state fermentation by isolated Bacillus

Aim: Investigation of mixture‐design impact on glutaminase production by isolated Bacillus sp. Methods and Results: An augmented simplex centroid design was used to optimize a three (wheat bran, Bengal gram husk and palm seed fibre) component mixture for glutaminase production. Selected substrate materials showed impact on glutaminase production values at individual level by Bengal gram husk [2789 U gds^?1 (gram dry substrate] and in two‐level combination with wheat bran and Bengal gram husk (maximum of 3300 U gds^?1). Conclusion: Bengal gram husk is the most suitable substrate medium for glutaminase production by Bacillus sp. Maximum glutaminase production is achieved using solid‐substrate mixture at two‐level combinations in the ratio of 66 : 34 for Bengal gram husk and wheat bran, respectively. Significance and Impact of the Study: The present study has significance in large‐scale production of glutaminase at commercial level with the use of multi‐substrate rather than single‐substrate/support material.     

On the two components of pyridoxal 5'-phosphate synthase from Bacillus subtilis

Vitamin B6 is an essential nutrient in the human diet. It can act as a co-enzyme for numerous metabolic enzymes and has recently been shown to be a potent antioxidant. Plants and microorganisms have the ability to make the compound. Yet, studies of vitamin B6 biosynthesis have been mainly restricted to Escherichia coli, where the vitamin is synthesized from 1-deoxy-d -xylulose 5-phosphate and 4-phosphohydroxy-l-threonine. Recently, a novel pathway for its synthesis has been discovered, involving two genes (PDX1 and PDX2) neither of which is homologous to any of those participating in the E. coli pathway. In Bacillus subtilis, YaaD and YaaE represent the PDX1 and PDX2 homolog, respectively. The two proteins form a complex that functions as a glutamine amidotransferase, with YaaE as the glutaminase domain and YaaD as the acceptor and pyridoxal 5'-phosphate (PLP) synthesis domain. In this report we corroborate a recent report on the identification of the substrates of YaaD and provide unequivocal proof of the identity of the reaction product. We show that both the glutaminase and synthase reactions are dependent on the respective protein partner. The synthase reaction can also utilize an external ammonium source but, in contrast to other glutamine amidotransferases, is dependent on YaaE under certain conditions. Furthermore, we report on the detailed characterization of the inhibition of the glutaminase domain, and thus PLP synthesis, by the glutamine analog acivicin. Employing pull-out assays and native-PAGE, we provide evidence for the dissociation of the bi-enzyme complex under these conditions. The results are discussed in light of the nature of the interaction of the two components of the enzyme complex.     

Adaptive ammoniagenesis in chronic renal failure.

The role of gamma-glutamyltransferase (gamma-GT) in renal ammoniagenesis, glutamine (Gln), and glutathione (GSH) utilization was evaluated in the intact functioning rat kidney of subtotal nephrectomy (SNX) model of chronic renal failure (CRF). NH4+ derived from extracellular gamma-GT hydrolysis of Gln and GSH was differentiated from the intramitochondrial phosphate-dependent glutaminase by using acivicin, a gamma-GT-specific inhibitor. In the control (C) group Gln extraction accounted for 61% of total NH4+ production (sum of renal venous and urinary NH4+), but only 41% in SNX group. In the SNX group GSH extraction accounted for 10% of total NH4+ production, but only 1% in the C group. Acivicin inhibited 44% and 33% of total NH4+ production in SNX and C group respectively, as compared to baseline before acivicin. In CRF, gamma-GT a key enzyme of the gamma-glutamyl cycle plays a significant role in adaptive ammoniagenesis.     

Distribution of hepatic glutaminase activity and mRNA in perivenous and periportal rat hepatocytes.

Perivenous and periportal hepatocytes were isolated by the digitonin/collagenase perfusion technique. The specific activity of phosphate-activated glutaminase was 2.33-fold higher in periportal cells than in perivenous cells. Similarly, the relative abundance of glutaminase mRNA was 2.6-fold higher in samples from periportal cells. The distribution of glutaminase activity and mRNA was compared with those for glutamine synthetase (predominantly perivenous) and phosphoenolpyruvate carboxykinase (predominantly periportal). The results suggest that phosphate-activated glutaminase is predominantly expressed in the periportal zone of the liver acinus.     

Enhanced anti-proliferative effects of combinatorial treatment of delta-tocopherol and resveratrol in human HMC-1 cells

The tocopherols (alpha, beta-, gamma-, and delta-tocopherol) and resveratrol are phytochemicals with alleged beneficial effects against atherosclerosis, vascular diseases and different cancers. They both can act as antioxidants, but they also modulate signal transduction and gene expression by non-antioxidant mechanisms. Here we wanted to determine whether the combined treatment of mast cells with the two compounds inhibits cell proliferation more efficiently when compared to individual treatments. Both compounds inhibit HMC-1 mastocytoma cell proliferation and reduce the activity of Protein Kinase B (PKB/Akt) by inhibiting its Ser473-phosphorylation. The combination of 50 microM delta-tocopherol and 50 microM resveratrol inhibits proliferation of HMC-1 cells more efficiently when compared to single treatments. In line with this, PKB Ser473-phosphorylation is inhibited best by delta-tocopherol and resveratrol combinatory treatment. Resveratrol acts more efficiently as an inhibitor of PKB phosphorylation than alpha-, beta-, gamma-tocopherols, whereas delta-tocopherol shows a stronger inhibition possibly as a result of its apoptotic secondary effects. Our data suggest that delta-tocopherol and resveratrol can act additively in reducing cell proliferation and PKB phosphorylation. The combination of phytochemicals with relatively broad specificity on enzymes involved in signal transduction and gene expression may increase their activity in disease prevention by modulating several different molecular targets.     

Antisense glutaminase inhibition decreases glutathione antioxidant capacity and increases apoptosis in Ehrlich ascitic tumour cells.

Glutamine is an essential amino acid in cancer cells and is required for the growth of many other cell types. Glutaminase activity is positively correlated with malignancy in tumours and with growth rate in normal cells. In the present work, Ehrlich ascites tumour cells, and their derivative, 0.28AS-2 cells, expressing antisense glutaminase mRNA, were assayed for apoptosis induced by methotrexate and hydrogen peroxide. It is shown that Ehrlich ascites tumour cells, expressing antisense mRNA for glutaminase, contain lower levels of glutathione than normal ascites cells. In addition, 0.28AS-2 cells contain a higher number of apoptotic cells and are more sensitive to both methotrexate and hydrogen peroxide toxicity than normal cells. Taken together, these results provide insights into the role of glutaminase in apoptosis by demonstrating that the expression of antisense mRNA for glutaminase alters apoptosis and glutathione antioxidant capacity.     

The role of glutamine and glucose analogues in metabolic inhibition of human myeloid leukaemia In vitro

• 1.1.Glutamine analogues l-[alphaS,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) and 6-diazo-5-oxo-l-norleucine (DON) have been shown to possess cytotoxic activity against a wide variety of animal and human xenografted solid tumours, however their potential in man has been limited by toxicity.
• 2.2.We have analysed the effects of acivicin and DON on glutamine utilization to determine whether the reason for the disappointing therapeutic profile is solely due to the inefficient inhibition of glutamine metabolism.
• 3.3.Human myeloid leukaemic cells treated with acivicin inhibited ribonucleotide biosynthesis but not energy production via glutaminolysis and had little effect on viability, whereas treatment with DON inhibited both ribonucleotide biosynthesis and glutamine oxidation and resulted in reduced viability.
• 4.4.Treatment of the myeloid leukaemic cells with the glucose analogue 2-deoxy-d-glucose in addition to DON potentiated the inhibition of de novo nucleotide biosynthesis, glutaminolysis and glycolysis, and caused a further reduction in cell viability.
• 5.5.These results provide further support for the essential role of glutamine in cellular metabolism, and indicate that use of the glutamine analogue DON in the treatment of acute myeloid leukaemia may be more clinically effective if used in combination with 2-deoxy-d-glucose.

     

The C-terminus of human glutaminase L mediates association with PDZ domain-containing proteins

The enzyme glutaminase in brain is responsible for the synthesis of neurotransmitter glutamate. We used the two-hybrid genetic selection system in yeast to look for interactors of glutaminase in human brain. We have identified two proteins containing PDZ domains, alpha1-syntrophin and a glutaminase-interacting protein, named GIP, that showed association with human glutaminase L, as deduced from specificity test of the two-hybrid system. The complete GIP cDNA clone has 1315 nucleotides with a 372-base open reading frame encoding a 124-amino acids protein. Glutaminase associates with both PDZ proteins through its C-terminal end; mutagenesis of single amino acids revealed the sequence -ESXV as essential for the interaction. These data suggest the possibility that PDZ domain-containing proteins are involved in the regulation of glutaminase in brain.     

Properties of phosphate activated glutaminase in astrocytes cultured from mouse brain

Astrocytes in primary cultures contain a relatively high activity, of phosphate activated glutaminase, although it is significantly lower than that of synaptosomal enriched preparations. The relatively high glutaminase activity in the astrocytes appears not to be caused by substrate induction, since a 10-fold variation in the glutamine concentration of the culture medium does not affect the activity. Of the reaction products, only glutamate inhibits astrocytic glutaminase whereas that of synaptosomal enriched preparations is inhibited by both glutamate and ammonia. Similar to the synaptosomal enzyme, glutaminase in astrocytes is inhibited about 50% by N-ethylmaleimide, indicating N-ethylmaleimide-sensitive and-insensitive compartments of the enzyme. Calcium activates glutaminase in astrocytes as in synaptosomes, by promoting phosphate activation. Except for the lower activity and the lack of effect of ammonia, the properties of the astroglial glutaminase has been found to be no different from that of the synaptosomal one. The relatively unrestrained astroglial glutaminase may, however, argue against the concept of a glutamine cycle operating in a stoichiometric manner.Abbreviations NEM N-ethylmaleimide - PAG Phosphate-activated glutaminase - PMB p-mercuribenzoate